Contrast agents

ABSTRACT

The invention relates to ultrasound contrast agents comprising vesicles comprising a protein capable of formation of gas-containing vesicles, wherein the vesicles contain gas which comprises sulphur hexafluoride or a low molecular weight fluorinated hydrocarbon. These contrast agents exhibit stability in vivo upon administration so as to permit ultrasound visualization while allowing rapid subsequent elimination from the system.

[0001] This invention relates to novel contrast agents, moreparticularly to new gas-containing or gas-generating contrast agents ofuse in diagnostic ultrasonic imaging.

[0002] It is well known that ultrasonic imaging comprises a potentiallyvaluable diagnostic tool, for example, in studies of the vascularsystem, particularly in cardiography, and of tissue microvasculature. Avariety of contrast agents has been proposed to enhance the acousticimages so obtained, including suspensions of solid particles, emulsifiedliquid droplets, gas bubbles and encapsulated gases or liquids. It isgenerally accepted that low density contrast agents which are easilycompressible are particularly efficient in terms of the acousticbackscatter they generate, and considerable interest has therefore beenshown in the preparation of gas-containing and gas-generating systems.

[0003] Initial studies involving free gas bubbles generated in vivo byintracardiac injection of physiologically acceptable substances havedemonstrated the potential efficiency of such bubbles as contrast agentsin echocardiography; such techniques are severely limited in practice,however, by the short lifetime of the free bubbles. Interest hasaccordingly been shown in methods of stabilising gas bubbles forechocardiography and other ultrasonic studies, for example usingemulsifiers, oils, thickeners or sugars.

[0004] WO 80/02365 discloses the use of gelatin encapsulated gasmicrobubbles for enhancing ultrasonic images. Such microbubbles do not,however, exhibit adequate stability at the dimensions preferred for usein echocardiography (1-10 μm) in view of the extreme thinness of theencapsulating coating.

[0005] EP-A-0327490 discloses, inter alia, ultrasonic contrast agentscomprising a microparticulate synthetic biodegradable polymer (e.g. apolyester of a hydroxy carbonic acid, a polyalkyl cyanoacrylate, apolyamino acid, a polyamide, a polyacrylated saccharide or apolyorthoester) containing a gas or volatile fluid (i.e. having aboiling point below 60° C.) in free or bonded form. Emulsifiers may beemployed as stabilisers in the preparation of such agents, but suchemulsifiers do not chemically interact with the polymer.

[0006] U.S. Pat. No. 4,774,958 discloses the use of microbubbledispersions stabilised by encapsulation in denatured protein, e.g. humanserum albumin (HSA). Such systems permit the production of microbubblesystems having a size of e.g. 2-5 μm but still do not permit efficientvisualization of the left heart and myocardium.

[0007] Other ultrasound contrast agents using proteins as encapsulatingagents have been described in the literature, for example in EP 0359 246(Molecular Biosystems), U.S. Pat. No. 4,832,941 (Max-PlanckGessellschaft), U.S. Pat. No. 4,844,882 (Molecular Biosystems), WO84/02838 (Feinstein), U.S. Pat. No. 4,572,203 (Feinstein), EP 0077 752(Schering), U.S. Pat. No. 4,747,610 (The Regents of the University ofCalifornia), WO 80/02365 (Rasor), U.S. Pat. No. 4,774,958 (Feinstein),U.S. Pat. No. 4,718,433 (Feinstein), EP 0224 934 (Feinstein).

[0008] The only protein-based ultrasound contrast agent under commercialdevelopment consists of a suspension of gas-filled albumin, Albunex®,prepared by sonication of a solution of albumin.

[0009] Albumin based ultrasound contrast agents are described in thefollowing publications:

[0010] Feinstein et al. in Circulation 78S, 565 (1988), Reisner et al.in Circulation 78S, 565 (1988), Dick et al. in Circulation 78S, 565(1988), Armstrong et al. in Circulation 78S, 565 (1988), Desir et al. inCirculation 78S, 566 (1988), Heidenreich et al. in Circulation 78S, 566(1988), Keller et al. in Circulation 78S, 567 (1988), Barnhart et al. inContrast Media Research (1989), Silverman et al. in Circulation 80S, 369(1989), Silverman et al. in Circulation 80S, 349 (1989), Segar et al. inClin.Res. 37, 294 (1989), Heidenreich et al. in Circulation 80S, 370(1989), Reiser et al. in Circulation 80S, 370 (1989), Heidenreich et al.in Circulation 80S, 566 (1989), Shandas et al. in Circulation 82, 95(1990), Geny et al. in Circulation 82, 95 (1990), Ten-Cate et al. in EurHeart J. 19, 389 (1989), Feinstein et al. in Echocardiography 6, 27(1989), Zotz et al. in Eur Heart J. 11, 261 (1990), Ten-cate et al. inEur Heart J. 11, 261 (1990), Barnhart et al. in Invest Radiol 25S, 162(1990), Keller et al. in J. Am Soc Echo 2, 48 (1989), Bleeker et al. inJ. Acoust Soc Am 87, 1792 (1990), Feinstein et al. in J. Am. Coll.Cardiol 16, 316 (1990), Kaul et al. in J. Am Coll. Cardiol 15, 195(1990), Bleeker et al in J. Ultrasound Med 9, 461 (1990), Hilpert et al.in Radiology 173, 361 (1989), and Shapiro et al. in J. Am. Coll. 16,1603 (1990).

[0011] However, as indicated above, ultrasound c ntrast agents based ongas-filled protein microspheres are unstable in vivo, and there is roomfor improvement of such products. Segar et al. have, in Advances inEchocardiography (September 21-22, 1989), concluded that batch, mixingpressure, mixing time and medium all affect the left atrium contrastwith such protein based products.

[0012] Feinstein et al. have in J. Am. Coll. Cardiol 16, 316 (1990)published that irrespective of dose group, a cavity opacification withalbumin microspheres was seen in the right ventricle in 88% of theinjections and in the left ventricle in 63% of the injections. Shandaset al. have in Circulation 82, 95 (1990) raised questions about thepressure related stability of gas filled albumin microspheres andShapiro et al. have recently published in J. Am. Coll. Cardiol 16, 1603(1990) lack of ultrasound myocardial contrast enhancement afteradministration of sonicated albumin.

[0013] Feinstein has in EP 0224 934 on page 4,8 and claim 9, U.S. Pat.No. 4,718,433 columns 3 and 5 and U.S. Pat. No. 4,774,958 columns 3 and5 suggested chemical denaturation to stabilize albumin gas bubbles:

[0014] “The microbubbles formed from 5% albumin may, in the alternative,be stabilized to form a commercially, clinically usable contrast agentby treatment with various chemical agents which chemically denature, or“fix”, the protein, and derivatives thereof. Chemical denaturation ofthe protein (or derivatives) may be accomplished bye. either binding theprotein with a protein-reactive aldehyde, such as glutaraldehyde. Forthe latter procedure of stabilizing the invented microbubble contrastagent, the microbubbles may be reacted with 0.25 grams of 50% aqueousglutaraldehyde per gram of protein at pH4:5 for 6 hours. The treatedcontrast agent is then gently and extensively washed to remove as muchof the unreacted glutaraldehyde as possible.”

[0015] Various denaturing chemicals or crosslinking agents for proteinshave been described in the literature. (See for example Methods Enzymol172, 584 (1989) and Chemical Reagents for Protein Modification, VolumeII, page 123, CRC Press Inc.)

[0016] However it is important that any contrast agent should be rapidlyeliminated from the subject in a short term after use, e.g. preferablyhaving a half life of not more than 48 hours. Crosslinking byglutaraldehyde or formaldehyde may not always be effective in providingan adequate balance between stability during ultrasound visualisationand rapid elimination. The protein itself, being human serum albumin, isnot rapidly degraded by vascular enzymes and reagents such asglutaraldehyde do not form readily biodegradable bonds with the protein.

[0017] The present invention is based on the concept of crosslinking theprotein shells of microbubbles to introduce biodegradable linkinggroups, thus providing ultrasound contrast agents with adequatestability for the duration of ultrasound visualisation but sufficientbiodegradability to permit rapid elimination subsequently.

[0018] According to the present invention, therefore, we provideultrasound contrast agents comprising microbubbles of gas or a gasprecursor encapsulated in a shell of protein crosslinked withbiodegradable crosslinking groupings.

[0019] Biodegradable linkages which may be used include amide, imide,imine, ester, anhydride, acetal, carbamate, carbonate, carbonate esterand disulphide groups. At least one such group should preferably bepresent in the crosslinking grouping. In general, any esters will bebiodegradable particularly those containing the grouping —CO.O— or—O.CO.O—. One particularly useful class of biodegradable ester groupingshas the structure

—(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n)—

[0020] (where Y and Z, which may be the same or different, are —O—,—S—or —NR³—; the symbols n, which may be the same or different, are zeroor 1; R¹ and R², which may be the same or different, are hydrogen atomsor carbon-attached monovalent groups or together represent acarbon-attached divalent organic group; and R³ is a hydrogen atom or anorganic group. Y and Z are preferably —O—. Such groups generally degradeto eliminate a compound R¹R²CO and either form carboxyl groups on theresidue or, in the case of carbonate esters, may eliminate carbondioxide to form hydroxyl groups on the residue.

[0021] R¹, R² and R³ may each be a hydrocarbyl or heterocyclic group,for example having 1-20 carbon atoms, e.g. an alkyl or alkenyl group(preferably having up to 10 carbon atoms), a cycloalkyl group(preferably having up to 10 carbon atoms), an aralkyl group (preferablyhaving up to 20 carbon atoms), an acyl group (preferably having up to 20carbon atoms) or a heterocyclic group having up to 20 carbon atoms andone or more heteroatoms selected from O,S and N; such a hydrocarbyl orheterocyclic grouping may carry one or more functional groups such ashalogen atoms or groups of the formulae —NR⁴R⁵,—CONR⁴R⁵,—OR⁶,—SR⁶ and—COOR⁷, where R⁴ and R⁵, which may be the same or different, arehydrogen atoms, acyl groups or hydrocarbyl groups as defined for R¹ andR²; R⁶ is a hydrogen atom or an acyl group or a group as defined for R¹or R² and R⁷ is a hydrogen atom or a group as defined for R¹ or R²;where R¹ and R² r present a divalent grouping, this may for example bean alkylene or alkenylene group (preferably having up to 10 carbonatoms) which may carry one or more functional groups as defined above.In general R¹ and R² are preferably hydrogen or small groups such asC₁₋₄ alkyl groups.

[0022] The protein component can be any protein or derivative thereofincluding polyamino acids. Albumin, gelatin and γ-globulin arerepresentative compounds. The protein, for instance albumin, can beobtained from biological sources, for example from human or animalblood, or produced by a lower organism using recombinant technology. Atypical method for preparation of human serum albumin by fermentation isdescribed in WO 9002808 (Delta Biotechnology Ltd.).

[0023] According to a further feature of the invention, we provide aprocess for the preparation of microbubble ultrasound contrast agents inwhich a gas or a gas precursor is encapsulated in a protein which iscrosslinked with biodegradable crosslinking groups.

[0024] The crosslinking of the protein can be effected before, during orafter encapsulation. It is preferred to encapsulate, e.g. by formingmicrobubbles, first and to effect crosslinking subsequently.

[0025] The crosslinking agent may be a compound of the formula (I)

A¹-X-A²   (I)

[0026] where X is a linking group containing one or more biodegradablelinkages and the groups A¹ and A², which may be the same or different,are functional groups reactive with proteins.

[0027] The group X may carry further groups reactive with proteins toprovide an even gr ater degree of crosslinking.

[0028] Preferably, the group X should have a chain length of not morethan 30 atoms.

[0029] The group X may thus be of the form

—R⁸-E-R⁹—

[0030] where R⁸ and R⁹ , which may be the same or different, aredivalent organic groups, for example alkylene or alkylidene groupshaving 1-12 carbon atoms, which may carry groups reactive with proteinsand/or further inert groups, and the group E is an ester grouping, forexample of the formula —O.CO—, —O.CO.O— or—(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n)— as defined above.

[0031] Crosslinking agents of the formula

A¹.R⁸.(Y)_(n).CO.O.C(R¹R²).O.CO.(Z)_(n).R⁹.A²

[0032] where A¹, A², R¹, R², R⁸, R⁹, n, Y and Z have the above meaningsmay be prepared by reaction of an acid of the formulaA¹.R⁸.(Y)_(n).CO.OH or a form thereof in which A¹ and any other reactivegroups are protected (or a functional derivative thereof) with acompound of the formula L¹.C(R¹R²).L² where L¹ is a leaving group suchas a halogen atom or mesyloxy or tosyloxy and L² is a group as definedfor L¹ (giving a symmetrical di-ester) or a group of the formula—O.CO.(Z)_(n).R⁹.A² or a protected form thereof, if necessary followedby deprotection. The functional derivative of the acid may for examplebe a salt, e.g. the potassium salt. The reaction will normally becarried out in solution, for example in a polar solvent such asdimethylformamide. Protecting groups for A¹ and A² may be thoseconventional in the art. Preferred protecting groups for aldehydesinclude acetals, e.g. cyclic acetals such as dioxolan.

[0033] The compound L¹.C(R¹R²).O.CO.(Z)_(n).R⁹.A², where L¹ is halogen,may be prepared from R¹R².CO by r action witha compound of the formulaHal.CO.(Z)_(n).R⁹.A² (where Hal represents a halogen atom) in thepresence of a base such as pyridine.

[0034] Apart from aldehyde groups, which are preferred, the groups A¹and A² may be activated carboxyl groups, such as N-hydroxysuccinimidylgroups (especially water solubility-enhanced sulphonatedN-hydroxysuccinimidyl derivatives), imidoesters, halo-nitroaryl groups,nitrene precursor groups such as azidophenyl, carbene precursor groups,ketone groups, isothiocyanate groups etc.

[0035] Any biocompatible gas may be employed in the contrast agents ofthe invention, for example air, nitrogen, oxygen, hydrogen, nitrousoxide, carbon dioxide, helium, argon, sulphur hexafluoride and lowmolecular weight optionally fluorinated hydrocarbons such as methane,acetylene or carbon tetrafluoride. The gas may be free within themicrobubble or may be trapped or entrained within a containingsubstance. The term “gas” as used herein includes any substance in thegaseous form at 37° C.

[0036] Gas precursors include carbonates and bicarbonates, e.g. sodiumor ammonium bicarbonate and aminomalonate esters.

[0037] For applications in echocardiography, in order to permit freepassage through the pulmonary system and to achieve resonance with thepreferred imaging frequency of about 0.1-15 MHz, it may be convenient toemploy microbubbles having an average size of 0.1-10 μm, e.g. 1-7 μm.Substantially larger bubbles, e.g. with average sizes of up to 500 μm,may however be useful in other applications, for examplegastrointestinal imaging or investigations of the uterus or Fallopiantubes.

[0038] As indicated above the microbubbles may be stabilised byincorporation of particulate material together with the encapsulatedgas. Such particles include, for example, silica and iron oxide. Thepreferred particle size for such stabilising particles is in the range 1to 500 nm, depending on the size of the microbubbles. The particlesshould be such that they are only partially wetted by the fluid mediumused to disperse the micelles, i.e. the contact angle between thematerial of the particles and the fluid should be about 90 degrees.

[0039] The stabilising particles may carry functional groups which willinteract with the protein to form covalent or other linkages. Colloidalsilica particles may have a particle size in the range 5-50 nm and maycarry silanol groups on the surface which are capable of interactionwith the protein by hydrogen bonding or by forming covalent bond.

[0040] The protein may stabilize the gas or gas precursor by forming amonolayer at the interface between the liquid medium and the gas or gasprecursor system, or by forming vesicles consisting of one or morebilayers containing the gas or gas precursor.

[0041] The stabilisation of the system by monolayers or the formation ofthe vesicles may be activated, as fully described in the literature, bysonication or even shaking of the protein material mixture in theappropriate medium, or the vesicles may be formed by any conventionalliposome/vesicle-forming principle.

[0042] The stabilized microbubbles may be dried or freeze-dried or thenon-aqueous phase may be evaporated. The resulting dried system may beresuspended in any physiological acceptable solvent such a saline orphosphate buffer, optionally using a suspending or emulsifying agent.

[0043] A gas entrapped system may be obtained by using a gas precursoror the gas itself may be entrapped. The gas may be entrapped into theamphiohile mixture simply by vigorously shaking the mixture in thepresence of air, i.e. creating a gas-in-liquid emulsion as described inU.S. Pat. No. 4,684,479. Another well established method, described i.e.in U.S. Pat. No. 4,774,958 for creating a gas-containing bubble is bysonication of the mixture in the presence of air. Another well knownmethod is passing the gas through a syringe into the mixture of theprotein and the liquid. As described in U.S. Pat. No. 3,900,420 themicrogas-emulsion may be created by using an apparatus for introducinggas rapidly into a fast-flowing liquid. A region of low pressure iscreated in a liquid containing the protein material. The gas is thenintroduced to the region of low pressure and the gas-in-liquid system isobtained by pumping the liquid through the system.

[0044] By using the principle of electrolysis it is possible to generatethe gas to be entrapped directly in a container containing the proteinmaterial. The electrolytes necessary for the electrolysis may even helpto further stabilize the protein material. An aqueous solutioncontaining electrolytes may generate hydrogen gas at the cathode andoxygen at the anode. The electrodes may be separated by a salt bridge.On adding hydrazine nitrogen gas may be generated at the anode. Usingthe Kolbe reaction, one may also generate CO₂ from carboxylic acidsusing electrolysis.

[0045] As described above, microbubbles may be obtained by formingliposomes or vesicles consisting of one or more bilayers. These vesiclesmay be formed at elevated pressure conditions in such a way that the gasis entrapped in the vesicles.

[0046] In one procedure according to the invention, encapsulation iseffected by agitation or sonication of the protein in an aqueous mediumto yield a protein foam which is dried and thereafter suspended in asolution of the crosslinking agent in a polar organic solvent (e.g. asulphoxide such as dimethyl sulphoxide) which is capable of wetting theprotein foam.

[0047] The following Examples are given by way of illustration only:

Preparation 1 Methylene bis (α-formylacetate)

[0048] The preparation of the starting material, the dioxolan-protectedaldehyde methyl α-formylacetate, is described by T. Hosokawa et al. J.Org. Chem. Soc. 52, (1987) 1758-1764. The protected aldehyde (6.0 g,3.75 mmol) is treated with a mixture of 2N aqueous potassium hydroxideand tetrahydrofuran 20:80 (v/v) at reflux for 8 hours. The pH isadjusted to 8 using diluted HCl, and the mixture is evaporated todryness. The solid is mixed with 100 ml freshly distilled and drieddimethylformamide, and after 30 minutes at 60° C. the undissolvedmaterial is filtered off. Diiodomethane (150 μl, 1.87 mmol) is addeddropwise during 5 minutes to the solution at 60° C. as described in WO89/00988 page 13 (NYCOMED AS). The precipitate is removed by filtrationafter stirring for 4 days, and the solvent removed at reduced pressure.The dioxolan protection is removed as described by P. A. Grieco et al.J. Am. Chem. Soc. 99, (1977) 5773-5780—the residue is dissolved intetrahydrofuran (60 ml), 5% aqueous HCl (20 ml) is added and the mixtureis stirred for 20 hours at ambient temperature. The reaction mixture isevaporated to dryness under reduced pressure to yield the titlecompound.

Preparation 2 Methylene Dimethacrylate

[0049] A solution of potassium hydroxide (1.00 M, 40.00 ml) is added tomethacrylic acid (3.44 g, 40.00 mmol) at 0° C. and the solution freezedried for 16 hours. Dry dimethylformamide (230 ml) is added and thesuspension heated to 60° C. under a dry nitrogen atmosphere.Diiodomethane (1.61 ml, 20.00 mmol) is added in two portions during 10min. and the reaction mixture left for 4 days at 60° C. The solvent isremoved under reduced pressure (0.05 mm Hg), before.diethyl ether (140ml), saturated acueous sodium hydrogen carbonate (50 ml) and water (50ml) are added. The aqueous layer is extracted with diethyl ether (6×60ml) and the combined ether exracts washed with water (4×50 ml), dried(MgSO₄), and evaporated to give 2.63 g (72%) of the title compound. ¹HNMR (60 MHz, CDCl): δ 1.97 (2×CH, m), 5.63 (2×H—C═, m), 5.88 (CH₂, s),6.18 (2×H—C═, m). IR (film, c=⁻¹): 2987 (w), 2962 (w), 2930 (w), 1732(str), 1638 (w), 1454 (w), 1315 (w), 1295 (w), 1158 (w), 1100 (str),1012 (m), 989 (m). This product may be used in accordance wizh theinvention, for example to crosslink acrylamide polymers.

Preparation 3 Methylene Diacrylate

[0050] A solution of potassium hydroxide (1.00 M, 40.00 ml) is added toacrylic acid (2.88 g, 40.00 mmol) at 0° C. and the solution freeze driedfor 16 hours. Dry dimethylformamide (200 ml) is added and the suspensionheated to 60° C. under a dry nitrogen atmosphere. Diiodomethane (1.61ml, 20.00 mmol) is added in two portions during 10 min. and the reactionmixture left for 4 days at 60° C. The solvent is removed under reducedpressure (0.05 mm Hg), before diethyl ether (140 ml), saturated aaueoussodium hydrogen carbonate (50 ml) and water (50 ml) are added. Theaqueous layer is extracted with diethyl ether (6×60 ml) and the combinedether extracts washed with water (4×50 ml), dried (MgSO₄), andevaporated to give 1.06 g (34%) of the title compound. ¹H NMR (60 MHz,CDCl₃): δ 5.81-6.61 (2×CH₂═CH—, m), 5.84 (CH₂, s). This product may beused in accordance with the invention, for example to crosslink acrylicacid and methyl acrylate polymers.

Preparation 4 Chloromethyl (2-methacryloyloxy)ethyl Carbonate

[0051] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (0.89 ml, 11.00 mmol) and 2-hydroxyethylmethacrylate (1.22 ml, 10.00 mmol) in dichloromethane (12 ml) at 0° C.under a dry nitrogen atmosphere. After 21 hours at 20° C. the reactionmixture is washed with hydrochloric acid (1.00 M, 10 ml), saturatedaqueous sodium hydrogen carbonate (10 ml) and water (10 ml). The organicphase is dried (MgSO₄) and the solvent evaporated under reduced pressure(10 mm Hg) to give 1.97 g (88%) of the title compound. ¹H NMR (60 MHz,CDCl₃): δ 1.88 (CH₃, d, J=2 Hz), 4.35 (O—CH₂—CH₂—O, m), 5.47 (H—C═, m),5.63 (CH₂—Cl, s), 6.00 (H—C═, m).

Preparation 5 (2-Methacryloyloxy)ethyl Methacryloyloxymethyl Carbonate

[0052] A solution of potassium hydroxide (1.00 M, 5.00 ml) is added tomethacrylic acid (0.43 g, 5.00 mmol) at, 0° C. and the solution freezedried during 16 hours. Dry dimethylformamide (50 ml) is added and to theresulting suspension is added chloromethyl (2-methacryloyloxy) ethylcarbonate (1.11 g, 5.00 mmol). 18-Crown-6 (0.066 g, 0.25 mmol) is addedas a catalyst and the reaction left under a dry nitrogen atmosphere.After 24 hours at 20° C. and 6 days at 4° C. the solvent is removedunder reduced pressure (0.05 mm Hg) and diethyl ether (30 ml) and water(20 ml) added. The aqueous layer is extracted with diethyl ether (3×20ml) and the combined ether extracts washed with water (20 ml), dried(MgSO₄) and evaporated to give 1.26 g (93%) of the title compound. ¹HNMR (60 MHz, CDCl₃): δ 1.97 (2×CH ₃, m), 4.38 (O—CH₂—CH₂—O, m), 5.53(2×H—C═, m), 5.77 (CH₂, s), 6.07 (2×H—C═, m).

Preparation 6 Ethylene bis(chloromethyl carbonate)

[0053] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (1.32 ml, 14.83 mmol) and ethylene glycol(0.28 ml, 5.00 mmol) in dichloromethane (10 ml) at 7° C. with goodstirring under a dry N₂ atmosphere. After 15 min. at 7° C. and 6 hoursat 20° C. the reaction mixture is transferred to a separating funnelwith the aid of dichloromethane (10 ml). The reaction mixture is washedwith hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodiumhydrogen carbonate (10 ml) and water (10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.12 g(90%) of the title product. ¹H NMR (300 MHz, CDCl₃): δ 4.48 (s,O—CH₂CH₂—O), 5.75 (s, 2×Cl—CH₂—O). ¹³C NMR (75 MHz, CDCl₃): δ 65.8(O—CH₂CH₂—O), 72.2 (2× Cl—CH₂—O), 153.0 (2×C═O).

Preparation 7 Bis(2-chloromethoxycarbonyloxyethyl)ether

[0054] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (1.32 ml, 14.83 mmol) and diethylene glycol(0.47 ml, 5.00 mmol) in dichloromethane (10 ml) at −7° C. with goodstirring under a dry N₂ atmosphere. After 10 min. at 7° C. and 6 hoursat 20° C. the reaction mixture is transferred to a separating funnelwith the aid of dichloromethane (10 ml). The reaction mixture is washedwith hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodiumhydrogen carbonate (10 ml) and water (10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure (10 mm Hg) togive 1.26 g (86%) title product. ¹H NMR (300 MHz, CDCl₃): δ 3.72 (m,2×CH₂—O), 4.34 (m, 2 × CH₂—O—C═O), 5.71 (s, 2×Cl—CH₂—O). ¹³C NMR (75MHz, CDCl₃): δ 67.6 (2×CH₂—O), 68.5 (2×CH₂—O—C═O), 72.1 (2 × Cl—CH₂—O),153.2 (2×C═O).

Preparation 8 1-Chloroethyl 2-methacryloyloxyethyl Carbonate

[0055] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution of1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and 2-hydroxyethylmethacrylate (1.22 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C.under a dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C.the reaction mixture is transferred to a separating funnel with the aidof dichloromethane (10 ml). The reaction mixture is washed, withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.76 g(74%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.85 (3 H, d, J=6Hz, CH ₃—CH), 1.96 (3 H,d, J=2 Hz, CH₃—C═), 5.55 (1 H, m, CH═), 6.10 (1H, m, CH═), 6.38 (1 H, k, J=6 Hz, CH—CH₃).

Preparation 9 Chloromethyl 4-acryloyloxybutyl Carbonate

[0056] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution ofchloromethyl chloroformate (0.98 ml, 11.00 mmol) and 4-hydroxybutylacrylate (1.38 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C. undera dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C. thereaction mixture is transferred to a separating funnel with the aid ofdichloromethane (10 ml). The reaction mixture is washed withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 1.76 g(74%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.82 (4 H, m,CH₂—CH₂), 4.27 (4 H, m, 2×CH₂—O), 5.77 (2 H, s, Cl—CH₂—O), 5.8-6.7 (3 H,m, CH═CH₂).

Preparation 10 1-Chloroethyl 4-acrloyloxybutyl Carbonate

[0057] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a solution of1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and 4-hydroxybutylacrylate (1.38 ml, 10.00 mmol) in dichloromethane (12 ml) at 3° C. undera dry N₂ atmosphere. After 15 min. at 3° C. and 17 hours at 20° C. thereaction mixture is transferred to a separating funnel with the aid ofdichloromethane (10 ml). The reaction mixture is washed withhydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium hydrogencarbonate (10 ml) and water (2×10 ml). The organic phase is dried(MgSO₄) and the solvent evaporated under reduced pressure to give 2.26 g(90%) of the title product. ¹H NMR (60 MHz, CDCl₃): δ 1.80 (4 H, m,CH₂—CH), 1.86 (3 H, d, J=5 Hz, CH₃), 4.24 (4 H, m, 2×CH₂—O), 5.7-6.6 (4H, m, CH═CH₂ and CH).

Preparation 11 1-Methacryloyloxyethyl 2-methacryloyloxyethyl Carbonate

[0058] 1-Chloroethyl 2-methacryloyloxyethyl carbonate (1.183 g, 5.00mmol) prepared as described in Preparation 8 is added to a suspension offreeze dried potassium methacrylate (0.683 g, 5.50 mmol) and 18-crown-6(0.066 g, 0.25 mmol) in dimethylformamide (50 ml) und r a dry N₂atmosphere. After 5 days at 20° C. the solvent is removed under reducedpressure and the residue dissolved by adding dichloromethane (60 ml) andwater (30 ml). After separating the phases the aqueous layer isextracted with dichloromethane (3×30 ml) and the combined organic phasewashed with saturated aqueous sodium hydrogen carbonate (50 ml). Theorganic phase is dried (MgSO₄) and the solvent removed under reducedpressure to give 1.10 g (77%) of the title product. ¹H NMR (60 MHz,CDCl₃): δ 1.63 (3 H, d, J=5 Hz, CH ₃—CH), 1.98 (6 H, s, 2 × CH₃), 4.42(4 H, s, O—CH₂—CH₂—O), 5.62 (2 H, m, CH═), 6.15 (2 H, m, CH═), 6.84 (1H, k, J=5 Hz, CH—CH₃).

Preparation 12 Acryloyloxymethyl 4-acryloyloxybutyl Carbonate

[0059] Chloromethyl 4-acryloyloxybutyl carbonate (1.183 g, 5.00 mmol)prepared as described in Preparation 9 is added to a suspension offreeze dried potassium acrylate (0.606 g, 5.50 mmol) and 18-crown-6(0.066 g, 0.25 mmol) in dimethylformamide (50 ml) under a dry N₂atmosphere. After 5 days at 20° C. the solvent is removed under reducedpressure and the residue dissolved by adding dichloromethane (60 ml) andwater (30 ml). After separating the phases the aqueous layer isextracted with dichloromethane (3×30 ml) and the combined organic phasewashed with saturated aqueous sodium hydrogen carbonate (50 ml). Theorganic phase is dried (MgSO₄) and the solvent removed under reducedpressure to give 1.24 g (91%) of the title product. ¹H NMR (60 MHz,CDCl₃): δ 1.82 (4 H, m, CH₂—CH₂), 4.23 (4 H, m, 2×CH₂—O), 5.88 (2 H, s,O—CH₂—O), 5.7-6.8 (6 H, 2×CH═CH₂).

Preparation 13 1-Acryloyloxyethyl 4-acryloyloxybutyl Carbonate

[0060] 1-Chloroethyl 4-acryloyloxybutyl carbonate (1.253 g, 5.00 mmol)prepared as described in Preparation 10 is added to a suspension offreeze dried potassium acrylate (0.606 g, 5.50 mmol) and 18-crown-6(0.066 g, 0.25 mmol) in dimethylformamide (50 ml) under a dry N₂atmosphere. After 5 days at 20° C. the solvent is removed under reducedpressure and the residue dissolved by adding dichloromethane (60 ml) andwater (30 ml). After separating the phases the aqueous layer isextracted with dichloromethane (3×30 ml) and the combined organic phasewashed with saturated aqueous sodium hydrogen carbonate (50 ml). Theorganic phase is dried (MgSO₄) and the solvent removed under reducedpressure to give 1.28 g (89%) of the title product. ¹H NMR (60 MHz,CDCl₃): δ 1.58 (3 H, d, J=5 Hz, CH ₃—CH), 1.80 (4 H, m, CH₂—CH₂), 4.24(4 H, m, 2×CH₂—O), 5.7-6.7 (6 H, m, 2×CH═CH₂), 6.87 (1 H, k, J=5 Hz,CH—CH₃).

Preparation 14

[0061] a) Methylene bis(3.3-dimethoxypropionate)

[0062] Cesium 3,3-dimethoxypropionate (19.95 g, 75 mmol) is added to dryDMF (1000 ml). Diiodomethane (10.04 g, 37.5 mmol) is added to thesuspension and the reaction mixture is stirred for 2 days at 60° C.under a dry N₂ atmosphere. DMF is removed under reduced pressure (0.01mmHg). Diethyl ether (500 ml) is added to the residue, which is thenwashed with saturated aqueous sodium hydrogen carbonate (250 ml). Theaqueous layer is extracted with diethyl ether (5×75 ml). The combinedether extracts are washed with water (2×100 ml), dried (MgSO₄) andevaporated to give 7.1 g (72%) product. ¹H NMR (300 MHz, CDCl₃): δ 2.61(CH₂, d), 3.26 (CH₃, s)

[0063] b) Methylene bis(3-methoxypropenoate)

[0064] Methylene bis(3,3-dimethoxypropionate) (14.01 g, 50 mmol)prepared as described in (a) above and a catalytic amount of p-toluenesulfonic acid is added to toluene (250 ml). The methanol is removed bywarming the reaction under an N₂ atmosphere. When the reaction iscomplete the toluene is distilled off under reduced pressure. Diethylether (250 ml) is added and the mixture is washed with saturated aqueoussodium hydrogen carbonate (5×50 ml) and water (3×50 ml). The organiclayer is dried (MgSO₄) before evaporation to give 8.52 g (79%) product.¹H NMR (300 MHz, CDCl₃): δ 3.65 (2×CH₃, s), 5.2 (2×CH, d), 5.8(O—CH₂—O), 7.65 (2×CH₂, d).

Preparation 15

[0065] a) Methylene bis(10-undecenoate

[0066] 10-Undecylenic acid (12.75 g, 75 mmol) is dissolved in 100 mlwater. Cesium carbonate (13.04 g, 40 mmol) is added to the mixture. Thewater is removed under reduced pressure and the salt dried for 2 hoursin vacuo. The cesium salt is mixed with 150 ml DMF and diiodomethane isadded to the solution. The reaction is stirred for 3 days at 60° C.under an N₂ atmosphere. DMF is then removed under reduced pressure. Theresidue is purified through silica gel with hexane/ ethyl acetate (8:2)as eluant. The solvent is evaporated to give 7.18 g (54%) product. ¹HNMR (300 MHz, CDCl₃): δ 1.2-1.4 (10 × CH₂, m), 1.6 (2×CH₂, m), 2.0(2×CH₂, m), 2.19 (2×CH₂, t), 4.9 (2×H₂ C═, m), 5.88 (O—CH₂—O, s), 5.9(2×HC═, m). ¹³C NMR (300 MHz, CDCl₃): δ 24.92-33.98 (8×CH₂), 79.04(O—CH₂—O), 114.18 (═CH₂), 139.11 (═CH), 172.48 (C═O).

[0067] b) Methylene bis(10-epoxyundecanoate)

[0068] Methylene bis(10-undecenoate) (8.8 g, 25 mmol) prepared asdescribed in (a) above is added under an N₂ atmosphere to methylenechloride and cooled to 0° C. Metachloroperbenzoic acid 55% (15.75 g, 50mmol) is added to methylene chloride (150 ml) and the organic layer isseparated and dried (MgSO₄). The metachloroperbenzoic acid is then addeddropwise to the diester. After completed addition the temperature isincreased to 25° C. After 5 hours the reaction is complete. The mixtureis washed with saturated aqueous sodium sulphite (75 ml) and saturatedaqueous sodium hydrogen carbonate (2×75 ml). The organic layer ispurified on neutral aluminium oxide. The solvent is removed underreduced pressure to yield 8.45 g (82%) product. ¹H NMR (300 MHz, CDCl₃):δ 1.2-1.7(14×CH₂, m), 2.35(2×CH₂CO,t), 2.45 (2×CH,q), 2.75 (2×CH,q),2.90 (2×CH,m), 5.75 (O—CH₂—O). ¹³C NMR (300 MHz, CDCl₃): δ24.58 (CH₂),25.99 (CH₂), 28.94 (CH₂), 29.09 (CH₂), 29.32 (2×CH₂), 32.45 (CH₂), 33.92(CH₂), 47.06 (CH₂—O), 52.36 (CH—O), 79.06 (O—CH₂—O), 172.2 (C═O).

Preparation 16 Methylene bis(4-epoxypentanoate)

[0069] Metachloroperbenzoic acid (15.68 g, 55%, 50 mmol) is dissolved inmethylene chloride (200 ml). Water is separated and the organic layer isdried (MgSO₄). The resulting metachloroperbenzoic acid solution is addeddropwise to methylene bis(4-pentenoate) (4.10 g, 19 mmol) dissolved inmethylene chloride (50 ml). The mixture is stirred at ambienttemperature under nitrogen for 12 hrs, whereafter the reaction mixtureis washed with saturated aqueous sodium bicarbonate solution (50 ml),water (50 ml), dried (MgSO₄) and evaporated to give 3.61 g (78%) of thetitle compound as a crystalline product. ¹H NMR (300 MHz, CDCl₃): δ1.70-1.85 (2×CH,m), 1.95-2.10 (2×CH,m), 2.50-2.55 (2×CH, 2×CH₂,m), 2.75(2×CH,t), 3.0 (2×CH,m), 5.8 (O—CH₂—O, s). ¹³C NMR (75 MHz, CDCl₃): δ 27(2×CH₂), 30 (2×CH₂), 47 (2×CH ₂), 51 (2×CH), 79.8 (O—CH₂—O), 171.8(2×C═O).

Preparation 17 Methylene bis(2-butenoate)

[0070] Vinylacetic acid (4.3 g, 50 mmol) is added to an aqueous cesiumcarbonate solution (50 ml). The mixture is stirred for 5 min. and thenevaporated, and the residue is dried under vacuum for 2 hrs. Theresulting cesium salt and diiodomethane are added to dimethylformamide(200 ml) and the mixture is stirred for 24 hrs. at 50° C. undernitrogen, whereafter th dimethylformamide is removed under reducedpressure. The residue is dissolved in diethyl ether (100 ml) and washedwith saturated aqueous sodium bicarbonate (25 ml) and water (25 ml). Theorganic layer is dried (MgSO₄) and evaporated to give 1.32 g (29%)product. ¹H NMR (300 MHz, CDCl₃): δ 1.9 (2×CH₂,m), 5.8-5.9 (2×CH,m), 5.9(OCH₂O,s), 7.0-7.1 (2×CH,m).

Preparation 18 Methylene bis(chloroacetate)

[0071] Chloroacetic anhydride (12.75 g, 75 mmol), paraformaldehyde (2.25g, 75 mmol) and conc. sulfuric acid (15 drops) are added to methylenechloride (15 ml). The mixture is stirred for 24 hrs. at 50° C. undernitrogen, whereafter the reaction mixture is extracted with saturatedaqueous potassium carbonate until carbon dioxide emission ends. Theorganic layer is dried (MgSO₄), evaporated to dryness and the residue isdistilled (80° C., 0.15 mmHg) to yield 10.2 g (57%) product. ¹H NMR (200MHz, CDCl₃): δ 4.1 (2×CH₂Cl,s), 5.9 (CH₂,s). ¹³C NMR (200 MHz, CDCl₃): δ41.1 (CH₂Cl), 81.4 (O—CH₂—O), 166.4 (CO).

Preparation 19 Methylene bis(4-oxopentanoate)

[0072] 4-Oxopentanoic acid (11.6 g, 100 mmol) is dissolved inacetonitrile (70 ml), and 1,8-diazabicyclo [5.4.0]undec-7-ene (15.25 g,100 mmol) diluted with acetonitrile (30 ml) is added. Diiodomethane(13.4 g, 50 mmol) is added in one batch, and the reaction mixture isrefluxed under a nitrogen atmosphere. After 2 hours, gas chromatographyindicates full consumption of diiodomethane. The solvent is removed invacuo and the residual brown oil is transferred to a separation funnelwith ethyl acetate (200 ml) and water (75 ml). The organic phase iswashed with 1M sodium bicarbonate (25 ml) and water (3×25 ml), driedover MgSO₄, and the solvent is removed in vacuo to yield the titlecompound (10 g). ¹H NMR: δ 2.19 (2×CH₂, s), 2.760-2.804 (2×CH₂, t),2.600-2.645 (2×CH₂, t), 3.735 (CH₂ bridge, s).

Preparation 20 Methylene bis(succinimidylazelate)

[0073] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.49g, 7.71 mmol) was added in portions to a stirred solution of methylenebis(hydrogen azelate) from Example 25 (1.00 g, 2.57 mmol) andN-hydroxysuccinimide (0.89 g, 7.71 mmol) in dry dimethylformamide atambient temperature. After 20 hours stirring, the reaction mixture waspoured into ice-water and the product precipitated as an oil. Thecolourless oil was dissolved in diethylether (50 ml), washed with water(3×10 ml) and dried over MgSO₄. The solvent was removed under reducedpressure and hexane (5 ml) was added to the oily product. After sevendays storage at 4° C. the oil had crystallized to a white, waxy solid.Yield: 1.50 g (69%). m.p.: 45-47° C. ¹³C NMR (75 MHz, CDCl₃) δ: 24.42,24.46, 25.59, 28.48, 28.63, 30.85, 33.82, 79.61, 168.6, 169.30, 172.34.

Preparation 21 Methylene bis(sulphosuccinimidylazelate) Sodium Salt

[0074] Methylene bis(hydrogen azelate) (0.38 g, 1 mmol),H-hydroxysuccinimide sodium salt (0.48 g, 2.2 mmol) anddicyclohexylcarbodiimide (0.45 g, 2.2. mmol) were dissolved indimethylformamide (10 ml). The suspension was stirred overnight at roomtemperature under an atmosphere of nitrogen. The reaction mixture wasfiltered and purified by reversed phase chromatography (RP-8) withwater/acetonitrile (1:1) as eluant to give the title compound.

Preparation 22

[0075] a) Methylene bis(10,11-dihydroxyundecanoate)

[0076] N-Methylmorpholine-N-oxide (13.5 g, 11 mmol) and methylenebis(10-undecenoate) from Preparation 15(b) (19 g, 5 mmol) were dissolvedin 400 ml of a mixture of tetrahydrofuran and water (3:1 v/v). Acatalytic amount of osmium tetroxide was added, and the solution stirredat ambient temperature for 20 hours. TLC indicated complete consumptionof the starting material. Excess sodium hydrogen sulphite and sodiumchloride were then added to the reaction mixture. The product wasextracted from the resulting mixture with ethyl acetate (400 ml) and thewater phase was washed with ethyl acetate (3×50 ml). The combinedorganic phases were dried and evaporated, and the product recrystallisedfrom tetrahydrofuran to yield 14.5 g (68%) of the product as a whitesolid. ¹³C NMR (45 MHz) CD₃OD:δ 24.6-34.0 (16×CH₂), 66.6 (2 ×CH₂OH),72.3 (2×CHOH), 79.2 (O—CH₂—O), 174.0 (2×C═O).

[0077] b) Methylene bis(10-oxodecanoate)

[0078] Methylene bis(10,11-dihydroxyundecanoate) (2.24 g, 5 mmol) wasdissolved in 150 ml tetrahydrfuran. Sodium metaperiodate (2.06 g, 10mmol) was dissolved in 150 ml water and added dropwise to thetetrahydrofuran solution. TLC indicated full consumption of the diolafter 60 minutes, whereupon sodium chloride was added to the reactionmixture until the two phases separated. The water phase was extractedwith diethyl ether(3×50 ml). The combined organic phases was dried withmagnesium sulphate and evaporated to give the title product as an oil,1.43 g (74%). ¹³C NMR (45 MHz) CDCl₃: δ 21.9-43.9 (16×CH₂), 79.1(O—CH₂—O), 173.0 (2×C═O), 202.6 (2×CHO).

EXAMPLE 1

[0079] 1. Gas-filled albumin microspheres are prepared according toEP-A-0359 246 and resuspend d to homogeneity by gentle rolling on a vialroller.

[0080] 2. 25 ml of the suspension are poured into a 25 ml separatingfunnel and left for 30 min. The bottom 20 ml are discarded.

[0081] 3. To the remaining 5 ml is added 20 ml of a phosphate buffer (20mM NaPO₄, pH 7.0), and the resulting suspension is transferred to a vialwith a cap septum.

[0082] 4. The vial is centrifuged upside down at 170×g for 5 min.

[0083] 5. The solution underneath the microsphere layer is withdrawnusing a syringe, and the microspheres are resuspended in 25 ml of thephosphate buffer by 10 min of gentle rolling.

[0084] 6. Points 4 and 5 are repeated twice.

[0085] 7. The resulting suspension is centrifuged as in point 4, and themicrospheres are resuspended in the phosphate buffer to a finalconcentration of about 5×10⁸ particles per ml.

[0086] 8. The crosslinker methylene bis(α-formylacetate), prepared asdescribed in Preparation 1, is added to the suspension, and thecrosslinking reaction is allowed to proceed for the desired time(usually 30-60 min) under gentle rolling.

[0087] 9. 1.5 M Tris-HCl-buffer (pH 8.8) is added to a finalconcentration of 0.25 M, and the suspension is rolled gently for 10 min.

[0088] 10. The vial is centrifuged as in point 4, and the solutionunderneath the microsphere layer is removed as in point 5.

[0089] 11. The microspheres are resuspended in phosphate buffer (samevolume as final volume in point 9), and the suspension is rolled for 10min.

[0090] 12. Points 10 and 11 are repeated twice.

[0091] 13. The resulting suspension is centrifuged as in point 4, andthe microspheres are resuspended in the phosphate buffer to a finalconcentration of about 5×10⁸ particles per ml.

[0092] 14. This final suspension of crosslinked gas/albumin microspheresis stored at 4° C.

EXAMPLE 2-22

[0093] The procedure of Example 1 is repeated using crosslinking agentsprepared as described in Preparations 2-22, except that dimethylsuplhoxide is used in place of phosphate buffer in the processing of thegas-filled albumin microspheres according to steps 3-7 and thecrosslinking agent is added in step 8 as a solution in dimethylsulphoxide.

[0094] The number and size distribution of the products are determinedby Coulter counter analysis.

1. Contrast agents for use in diagnostic ultrasound studies comprisingmicrobubbles of gas or a gas precursor encapsulated in a protein shellcharacterised in that the said protein is crosslinked with crosslinkinggroupings containing biodegradable linkages.
 2. Contrast agents asclaimed in claim 1 wherein the crosslinking groupings containbiodegradable linkages selected from amide, imide, imine, ester,anhydride, acetal, carbamate, carbonate, carbonate ester and disulphidegroups.
 3. Contrast agents as claimed in claim 2 wherein thecrosslinking groups contain biodegradable linkages of formula—(Y)_(n)—CO—O—C(R¹R²)—O—CO—(Z)_(n)— (where Y and Z, which may be thesame or different, are —O—, —S— or —NR³—; R¹ and R², which may be thesame or different, are hydrogen atoms or carbon-attached monovalentorganic groups or together represent a carbon-attached divalent organicgroup; R³ is a hydrogen atom or an organic group; and the symbols n,which may be the same or different, are zero or 1).
 4. Contrast agentsas claimed in any of the preceding claims wherein the protein isalbumin, gelatin or globulin.
 5. Contrast agents as claimed in claim 4wherein the protein is human serum albumin.
 6. Contrast agents asclaimed in any of the preceding claims further containing an inorganicparticulate stabiliser.
 7. A process for the preparation of a contrastagent as claimed in claim 1 which comprises encapsulating a gas or gasprecursor in a protein and crosslinking the protein with crosslinkinggroups containing biodegradable linkages before, curing or after saidencapsulation.
 8. A process as claimed in claim 7 wherein crosslinkingis effected after encapsulation.
 9. A process as claimed in claim 7 orclaim 8 wherein crosslinking is effected using a crosslinking agent offormula (I) A¹-X-A²  (I) (where X is a linking group containing one ormore biodegradable linkages as defined in claim 2 or claim 3 and A¹ andA², which may be the same or different, are functional groups reactivewith proteins).
 10. A process as claimed in claim 9 in which A¹ and A²are both aldehyde groups.
 11. A process as claimed in any of claims 8 to10, wherein encapsulation is effected by agitation or sonication of theprotein in an aqueous medium to yield a protein foam which is dried andthereafter suspended in a solution of the crosslinking agent in a polarorganic solvent.
 12. A process as claimed in claim 11 in which thecrosslinking agent is a compound of formula (I) as defined in claim 9 inwhich A¹ and A² are both O-linked sulphonated N-hydroxysuccinimidylresidues.